The completion of Human Genome Project and genome sequencing on other species has had an enormous impact on human healthcare, quality of life, food, environment, and other living organisms. Sequencing of many other species is still ongoing. Now one of the major tasks is to understand the function and products of genomes. For a long time, there was a belief that RNA function was limited to their involvement in protein synthesis including messenger RNA, ribosomal RNA and transfer RNA (mRNA, rRNA, and tRNA, respectively). Recently identified small non-coding RNA molecules were discovered to be actively involved in gene regulation by directly interacting with mRNAs and silencing the genes, commonly referred to as RNA interference (RNAi). These RNA molecules are further classified into microRNAs (miRNAs) and small interference RNAs (siRNAs) and so on.
Although miRNAs play important roles in the regulation of gene expression, effective techniques for the detection and quantitation of miRNA expression are lacking. To date, the principal methods used for quantitation of miRNAs are based on gel electrophoresis. The miRNAs are detected either by Northern blotting or by the presence of radioactive RNase-resistant duplexes. Moreover, transfer of small RNAs to filters can introduce problems with reproducibility of quantitation and is not typically amenable to high-throughput. Moreover, detection methods based on Rnase resistance require highly radioactive probes. An alternative approach involves cloning the miRNAs and then sequencing the inserts. While this approach may be suitable for discriminating single-base differences between closely related miRNA species, it is time consuming and laborious.
The miRNAs and siRNAs are classes of small non-coding RNA molecules that are widely expressed in many cells and organisms, plants as well as animals. The mature forms of these RNAs are generally small (˜22 nucleotides), the precursors of which are either hairpin or long double-stranded RNA (dsRNA) molecules transcribed from the genomes. These molecules are actively involved in gene regulation, RNAi and gene silencing, and important biological and pathological processes of cells. Profiling miRNA, siRNA and other types of small RNA expression in tissues and cells would greatly air understanding of these molecular functions and discovery of biomarkers for diagnostics and therapeutics.
Microarrays have been a powerful tool to profile gene expression, particularly mRNAs. However, profiling small RNAs on microarrays has not been validated. What is needed is an efficient way to analyze small RNAs in quantity.